Abstract：
CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.
摘要：
CRM197EK是白喉类毒素交叉反应物质-197（CRM197）的衍生物，具有两点突变（K51E和E148K），可改善其疫苗缀合物和药物递送的特性。先前的研究表明，在大肠杆菌中细胞内表达CRM197EK（E.大肠杆菌）宿主形成的包涵体需要复杂的纯化和重折叠步骤。通过使用N-月桂酰肌氨酸(sarkosyl)溶解包涵体可克服从包涵体纯化蛋白质。在这项工作中,重组CRM197EK(rCRM197EK)在大肠杆菌BL21(DE3)中表达为包涵体,然后使用sarkosyl溶解以形成可溶性rCRM197EK，而无需复性过程。此外，使用Ni-NTA柱纯化rCRM197EK，通过SDS-PAGE和Western印迹表征，并通过其DNase活性测定其生物活性。此外，使用表面等离子体共振（SPR）测量其与抗白喉毒素（DT）抗体的结合亲和力。结果表明，SDS-PAGE和Western印迹证实了用sarkosyl形式的可溶性rCRM197EK（61.61kDa）的溶解，收率为2.8mg/mL。rCRM197EK显示DNase活性，SPR分析显示，它可以与抗DT抗体相互作用，结合能为-9.2kcal/mol。
